Accumulating experimental evidence from studies examining normal and neoplastic development in the rat supports the existence of bipotential facultative liver stem cells (FLSC), which express the fetal isozyme of alpha-fetoprotein (AFP) in adult liver. The long-term goal of this research project is to identify, isolate and propagate these FLSC from the normal adult rat liver. Our initial experimental approach will employ a panel of monoclonal antibodies against different liver cell types in combination with immunomagnetic beads and fluorescence activated cell sorting (FACS) to obtain antigenically defined populations of nonparenchymal cells (NPC). These populations will be further characterized for AFP expression using in situ hybridization and reverse transcriptase polymerase chain reaction to detect AFP+ cells. Subsequently, we propose to infect and isolate AFP+ NPC using a replication deficient adenoviral vector containing "capture" genes driven by AFP transcriptional regulatory sequences. Although all cells will be infected in vitro, only those cells actively transcribing the AFP gene will express the recombinant capture genes. These positive cells can then be isolated by magnetic microparticles or by FACS. The advantages of using an adenoviral vector are; 1) the high efficiency of infection of most epithelial cell types, and 2) the adenovirus is not integrated into the genome of AFP+ cells. Therefore, when cells are propagated most will "kick out" the adenoviral vector. Thus transient viral infection should not alter the differentiation potential of the AFP+ NPC. The purity of each fraction can subsequently be confirmed and if necessary, the adenoviral infection can be repeated. Moreover, this concept can be adapted for other lineage markers for which the gene specific transcriptional regulatory units are known, thus enabling us to purify any cell population by this technique. Presently, culture conditions are available for the continuous propagation of bile duct and mesothelial cells. However, it may be necessary to develop culture conditions for the propagation of AFP+ cells. If this is the case, then we will investigate the effects of using Ito cell feeder layers, condition media or cocultures on the proliferation of the presumptive FLSC--since these mesenchymal cells produce a variety of growth factors in vivo which are involved in the proliferation of oval cells--e.g. the progeny of liver stem cells. Finally, the biological potential of AFP+ NPC populations will be defined using several transplantation model systems which may promote the differentiation of presumptive bipotential FLSC into hepatocytic or biliary lineages. If we successfully demonstrate that AFP+ NPC are indeed bipotential hepatic precursors, then our experimental approach will provide a powerful tool to isolate this cell population from normal and carcinogen treated animals.